Genome-wide characterization and expression profiling of Eucalyptus grandis HD-Zip gene family in response to salt and temperature stress

Background The HD-Zip transcription factors are unique to plants and play an essential role in plant growth, development and stress responses. The HD-Zip transcription factor family consists of a highly conserved homeodomain (HD) and a leucine zipper domain (LZ) domain. Although the HD-Zip gene family has been extensively studied in many plant species, a systematic study of the Eucalyptus HD-Zip family has not been reported until today. Here, we systematically identified 40 HD-Zip genes in Eucalyptus (Eucalyptus grandis). Besides, we comprehensively analyzed the HD-Zips of Eucalyptus by studying the homology, conserved protein regions, gene structure, 3D structure of the protein, location of the genes on the chromosomes and the expression level of the genes in different tissues. Results The HD-Zip family in Eucalyptus has four subfamilies, which is consistent with other plants such as Arabidopsis and rice. Moreover, genes that are in the same group tend to have similar exon-intron structures, motifs, and protein structures. Under salt stress and temperature stress, the Eucalyptus HD-Zip transcription factors show a differential expression pattern. Conclusions Our findings reveal the response of HD-Zip transcription factors under salt and temperature stresses, laying a foundation for future analysis of Eucalyptus HD-Zip transcription factors.

The homologous domain leucine zipper (HD-Zip) gene family is reported only in the plants and regulates plant-specific growth and development processes [7,8]. HD-Zip possesses a highly conserved homeodomain (HD) composed of 61 amino acids and a Leucine zipper (LZ) domain. The HD motif can specifically bind to DNA, whereas LZ serves as a dimerization motif [9]. HD-Zips have been identified and analyzed in various plant species, such as Medicago truncatula, grape (Vitis vinifera), rice (Oryza sativa), maize (Zea mays), potato (Solanum tuberosum), wheat (Triticum aestivum) and banana [10][11][12][13][14][15][16][17]. The HD-Zips are divided into four subfamilies: HD-Zip I, HD-Zip II, HD-Zip III, HD-Zip IV based on their gene structure, conserved sequences, cis-elements and biological functions [18,19]. Similarly, Eucalyptus also has four subfamilies, HD-Zip I to HD-Zip IV. Among them, the HD-Zip I contains both of the two basic motifs; however, the HD-Zip II, III and IV families possess other motifs in addition to HD and LZ motifs. 1) CPSCE (consisting of five conserved amino acid sequences of Cys, Pro, Ser, Cys, and Glu) exist in the HD-Zip II subfamily [20,21]. 2) In the HD-Zip III and IV subfamilies, a steroidogenic acute regulatory protein motif START (star-related lipid transfer), which is associated with lipid transfer, is found [22,23]. HD-Zip I and HD-Zip II proteins recognize a similar pseudopalindromic sequence CAAT(C/G)ATTG [7,20,21], while HD-Zip III and HD-Zip IV proteins recognize the sequences GTAAT(G/ C)ATTAC and TAAATG(C/T)A, respectively [22,24].
HD-Zip I proteins are reported for their function in plant light signal transduction [5], leaf and seed development [20,24], plant growth, de-yellowing and plant response to stress [1,21,25]. In Arabidopsis, when overexpressed, an HD-Zip I member ATHB12, results in big leaves and enlarged cells, displaying its role in leaf growth. While another HD-Zip I member, ATHB1 contributes to cell wall composition and elongation [18,26]. Besides, Federico et al. proved that Medicago truncatula HD-Zip I TF HB1 is required for lateral root development [16,18]. Moreover, HD-Zip I proteins also play a vital role during abiotic stress responses. For example, drought and abscisic acid strongly upregulate ATHB7 and ATHB12, that act as positive regulators of PP2C in Arabidopsis [27]. In sunflower, HaHB4 regulates tolerance against drought conditions through ethylenemediated aging [28]. Besides, overexpression of ZmHDZip10 and TaHDZipI-5 can improve plant tolerance to low temperature, drought, or salinity stress [29][30][31]. HD-Zip I family members also have a defensive role in combating biotic stresses. For example, the pepper HD-Zip I protein has a positive effect on increased tolerance against Ralstonia solanacearum [32].
HD-Zip II subfamily possesses a conserved domain CPSCE, which plays an essential role in mediating plant response to changes in light quality and shading [33,34], and abiotic stress responses [35]. Photochemical conditions mainly regulate their expression [36,37].
The structure of the HD-Zip III proteins is most complex among HD-Zips. The HD-Zip III family possesses the START domain, homeodomain-START associated domain (HD-SAD) and Met-Glu-Lys-Hi-Leu-Ala (MEKHLA) domain. The START domain is ABAresponsive, and the function of the HD-SAD domain is currently unknown. It is worth noting that the HD domain of HD-Zip III has two altered amino acid residues compared to the other subfamilies, and this change may be related to the unique MEKHLA domain [38]. The MEKHLA domain is a PAS-like domain associated with several chemical and physical stimuli [39]. HD-Zip III proteins not only participate in plant-specific photosynthetic processes but also inhibit the transcription [40], implying that HD-Zip III genes might play a role in transcription [40]. HD-Zip III proteins are also believed to be involved in plant embryo development [41], meristem formation [39], vascular development [42], and polar transport of auxin during plant development [10]. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate the gene expression [43]. miRNAs bind to complementary mRNA molecules and negatively regulate the expression of targets through slicing or translational repression [44]. The class III HD-Zip genes are reported to be post-transcriptionally regulated by the microRNAs miR165/166 [45,46]. In Arabidopsis developing roots, cross-talk of at least six different phytohormones dynamically regulates the spatio-temporal expression pattern of miR165/166 and HD-Zip III. Besides, HD-Zip III mediated root development is modulated transcriptionally through phytohormones and KANs, and post-transcriptionally by miR165/ 166 [47]. Class II and class III HD-Zips determine the correct patterning of upper and lower leaf tissues, and together repress miRNAs miR165/166, that ultimately regulates class III HD-Zips function. This three-way interaction maintains tissue identity balance during development, which helps in the development of a flat leaf in Arabidopsis [48,49]. Also, miR166g over-expression in Arabidopsis jabba-1D (jba-1D) mutant plant affects the transcripts of class III homeodomain-leucine zipper family genes [50].
The HD-Zip IV subfamily contains four conserved domains identical to the HD-Zip III subfamily, the HD, Zip, START and HD-SAD domains. However, HD-Zip IV lacks the MEKHLA domain [51,52]. The genes of this subfamily are expressed explicitly in the outer cell, epidermal, sub-epidermal cells of multiple species during biotic and abiotic stresses [53,54]. HD-Zip IV also plays a vital role in trichome formation, anthocyanin accumulation, lipid biosynthesis and transport [53][54][55][56].
Eucalyptus, a native plant of the southeastern coast of Australia, is a member of the genus Myrtaceae. In China, Eucalyptus is mainly planted in Guangxi province. It enjoys moist and fertile river loam soil and red soil weathered by basalt. Eucalyptus has high economic value due to its short growth cycle and other advantages. The leaves of Eucalyptus can be used to extract aromatic oils [57]. Besides, it can also be used as medicine due to its antiinflammatory, sterilizing and expectorant effects [58]. Eucalyptus wood has corrosion resistance characteristics, and it is widely used in such construction, papermaking and fuel industries. Also, it is commonly used in artificial afforestation around the world, and as a unique commercial tree species, Eucalyptus is planted on a large scale. However, Eucalyptus is often affected by abiotic stresses such as drought, low temperature, and salt stress during its growth, resulting in a decline in the yield [59][60][61]. Therefore, it is crucial to study and analyze the stress response-related genes of Eucalyptus to improve the excellent traits and breeding.
Here, we identified and classified the HD-Zip proteins of Eucalyptus, analyzed the relationship between the HD-Zip family and HD-Zips responses to temperature and salinity stress. These results provide the necessary basis for the functional characterization of the Eucalyptus HD-Zip family and a theoretical basis for the subsequent improvement of Eucalyptus varieties against stress.

Phylogenetic and evolutionary analysis of HD-Zip proteins in Eucalyptus
A total of 40 candidate HD-Zip genes were identified in Eucalyptus and named as EgHD-Zip 1-40. To further analyze the selected sequences, a phylogenetic tree consisting of the 40 sequences was constructed. Also, to better highlight the difference between Eucalyptus and other plants, Arabidopsis and rice were included in the evolutionary analysis. The results showed that Eucalyptus, like Arabidopsis and rice, also had 4 subfamilies of HD-Zip genes. Among them, the HD-Zip I subfamily has the maximum members (15 Nos), and the HD-Zip III subfamily has the least members (4 Nos) (Fig. 1, Supplementary Fig. S1, S2).
In the II subfamily, EgHD-Zip16 and AT1G70920 (ATHB18), EgHD-Zip40 and AT4G16780 (ATHB2) showed a high homology. Besides, in the subfamily I, EgHD-Zip36 had high homology with AT1G26960 (ATHB23). In the subfamily III, EgHD-Zip19 and AT1G52150 (ATHB15) were highly homologous, so it can be speculated that EgHD-Zip19 may also have a similar role in inducing the development of vascular bundles. Similarly, EgHD-Zip20 and AT4G32880 (ATHB8) were on the same branch, so it can be speculated that EgHD-Zip20 could also induce the development of vascular bundles. In subfamily IV, EgHD-Zip4 Fig. 1 Phylogenetic classification of HD-ZIP gene in Eucalyptus grandis, Arabidopsis thaliana and Oryza sativa. The phylogenetic tree was constructed using iQTREE software. The topological structure of the ML tree shows that the HD-Zip genes of Eucalyptus, Arabidopsis and rice can be divided into four subfamilies, namely EgHD-Zip I, EgHD-Zip II, EgHD-Zip III, EgHD-Zip IV. In the figure, yellow represents the HD-Zip I subfamily of Arabidopsis, rice and Eucalyptus, blue represents the HD-Zip II subfamily of Arabidopsis, rice and Eucalyptus, and green represents the HD-Zip III subtype of Arabidopsis, rice and Eucalyptus. The orange represents the HD-ZipI IV subfamily of Arabidopsis, rice and Eucalyptus had high homology with AT1G79840 (ATHB10), EgHD-Zip5 and AT4G00730 (ANL2), suggesting that EgHD-Zip4 may be involved in the epidermal cell differentiation process, and EgHD-Zip5 can affect flower development. The accumulation of glucoside in the leaf epidermis, and when the mutation occurs, it will inhibit the accumulation of anthocyanin. At the same time, it may also be involved in determining the identity of cells in the root [7].
As shown in Table 1, the isoelectric points of proteins from the second subfamily are more than 7, suggesting these proteins contain more basic amino acids. While the isoelectric point for the genes from other subfamilies, is generally less than 7, suggesting each protein contains more acidic amino acids. Besides, EgHD-Zip proteins are 91 to 848 amino acids long, with an average length of 430 amino acids, a molecular weight of 10.39 kDa to 93.01 kDa, and an average of 47.51 kDa. Subcellular localization analysis indicates that the Eucalyptus HD-Zip genes are all localized in the nucleus.

Analysis of EgHD-Zip gene structure and motif structure
Multiple Em for Motif Elicitation (MEME) and Gene Structure Display Server (GSDS) were used to understand the relationship between EgHD-Zip proteins and their structure. It is worth noting that all of the EgHD-Zip proteins possessed the conserved motif encoding the HD and LZ domains. Highly homologous members were composed of the same motif, indicating that HD-Zip proteins of the same subfamily could have similar functions. The results also suggest that all EgHD-Zip proteins except EgHD-Zip30 have motifs 1, 2 and 5. Motifs 1 and 2 represent the conserved motifs encoding the HD domain, and motif 5 represents the encoding LZ conserved motifs of the domain (Fig. 2). The motif 12 encoding the CPSCE domain is found in all members of the HD-Zip II subfamily. However, HD-Zip-N is less conservative in the HD-Zip II subfamily because it was not found in EgHD-Zip16, − 22, − 25, − 28, − 30, − 32, and − 34. Also, the motif of the START domain was found in subfamilies III and IV. The MEKHLA domain encoded by motif 8 and motif 24 is unique to the subfamily III and found only in 4 members ( Supplementary Fig. S3, S4).
The Gene Structure Display Server (GSDS) was used to analyze the structural diversity of the HD-Zip genes in Eucalyptus (Fig. 3). The results showed that the genetic structure of the 40 HD-Zip genes differed significantly in exon/intron arrangement and the number of introns. At the same time, the most relevant members in the same subfamily had similar exons/introns. The intron structure and the number of introns were consistent with the characteristics defined in the phylogenetic analysis described above. Also, the structure of the subfamily III genes was most sophisticated among the HD-Zip gene family, consistent with the motif analysis results. For example, the subfamily I and subfamily II HD-Zip genes contain 2 to 4 exons, the subfamily IV HD-Zip genes contain 8 to 11 exons, and the subfamily III HD-Zip genes contain 18 exons.
To obtain intron gain/loss information for all sister pairs, we also compared the intron/exon structure of genes clustered at the end branches of the phylogenetic tree. Among them, 5 pairs showed intron/exon structural changes, including HD-Zip6/− 10, HD-Zip3/− 5, HD-Zip7/− 11, HD-Zip35/− 37, and HD-Zip39/− 30 (Fig. 4), these changes only occurred in the subfamilies I, II, and IV. By comparing with neighboring genes, we found that HD-Zip7 and HD-Zip6 have obtained an intron during the evolutionary process, while HD-Zip35 has lost an intron (Fig. 4).

3D structure analysis of EgHD-Zip
Using the protein homology modeling method based on the Eucalyptus HD-Zip structure of the Swissmodel database, the structures of the members from four subfamilies of Eucalyptus HD-Zip were predicted. The Nand C-termini of the 3D structure of EgHD-Zip protein are shown in Fig. 4. The structure with the highest score was chosen as the optimal structure for the EgHD-Zip protein. The 3D structure of each subfamily has a significant difference, and the 3D structure of HD-Zip III and HD-Zip IV was most complicated. Consistent with previous studies, HD-Zip I has some conserved structures at the carboxyl-terminal region (CTR) and aminoterminal region (NTR) [62]. As the simulation analysis revealed, the four subfamilies have significant differences in genes at the protein structure level.

Chromosome localization of EgHD-Zip genes
MapChart software was used to determine the distribution of EgHD-Zip gene based on their position on 11 chromosomes in Eucalyptus. We found that the distribution of HD-Zip genes in Eucalyptus was uneven. Chromosome 10 had only one HD-Zip gene, while the remaining chromosomes had two to five genes (Fig. 5). There were four genes of the first subfamily on chromosome 9, and subfamily II was mainly distributed on chromosome 5. The genes of subfamily III and IV were scattered in the Eucalyptus chromosome, and subfamily IV was on number 1. There were 1 or 2 genes on chromosomes 4, 5, 6, and 8 (Fig. 5).

Expression profiles of Eucalyptus HD-Zip genes in various tissues
For this experiment, samples from mature leaves, phloem, young xylem, mature xylem, and shoot tip of Eucalyptus were selected, and an expression heat map of EgHD-Zip genes was generated using TBtools (Fig. 6, Supplementary  Fig. S5, S6, S7, S8). The analysis showed that in subfamily IV, except for EgHD-Zip4 and EgHD-Zip38, the expression levels of other genes in xylem and phloem were lower. In comparison, the expression level in stem tip and leaves were higher, indicating that this family may play a role in the growth and development of apical meristems and leaves. Also, the four genes of subfamily III were highly expressed in xylem and phloem, suggesting that the genes from subfamily  It is worth noting that three genes were highly expressed in vascular tissue. Among them, EgHD-Zip26 was highly expressed in young xylem tissues and may play a unique role in the formation and development of xylem. Besides, EgHD-Zip35 was highly expressed in mature xylem, and it could be related to the transport of hormones, water and nutrients in plants. Similarly, EgHD-Zip29 was also highly expressed in the phloem, suggesting that it may play an important role in the development of the phloem. In subfamily II, 67% of genes are related to the growth and development of vascular bundles and the transport of plant hormones. Interestingly, some genes had low or no expression in the selected samples, which does not mean that these genes are not essential. Instead, it may be expressed in other developmental stages. In general, we found each subfamily has its unique expression characteristics and thus plays a different role.

EgHD-Zip gene expression profiles in response to salt
The response of Eucalyptus to temperature and salt stress is more prominent. We found that the response trend of EgHD-Zip27 and EgHD-Zip37 was the same under 0 mmol/L NaCl treatment. At 0 mmol/L NaCl EgHD-Zip27 and EgHD-Zip37 showed a decrease after 6 h followed by an increase suggesting that the plant's biological clock could regulate the expression levels of EgHD-Zip27 and EgHD-Zip37. At 100 mmol/L NaCl treatment, the expression levels of EgHD-Zip27 and EgHD-Zip37 were much higher compared to 0 mmol/L NaCl treatment, and first showed an increasing trend, then a decrease followed by an increase. Though the expression levels of EgHD-Zip27 and EgHD-Zip37 were significantly decreased at 200 mmol/L NaCl treatment, yet it was higher compared to 0 mmol/L NaCl treatment. Taken together, it can be inferred that EgHD-Zip27 and EgHD-Zip37 have essential roles in coping with NaCl stress (Fig. 7).

EgHD-Zip gene expression profiles in response to temperature
At 4°C, the expression of both EgHD-Zip27 and EgHD-Zip37 genes was lower than that at 25°C, especially the expression of EgHD-Zip37 gene decreased by half at 4°C. At 40°C, the expression of EgHD-Zip37 was also reduced by half, so it can be speculated that EgHD-Zip37 is inhibited by high temperature and low temperature. The expression pattern of EgHD-Zip27 at 40°C was the same as at 25°C (Fig. 8).

Discussion
The growth and development of plants are an extremely complicated process. In this process, plants are subjected  [63,64]. HD-Zip proteins are plant-specific transcription factors that play a significant role in plant development and response against various stresses [8,18,24,65]. In this study, a comprehensive identification and analysis of EgHD-Zip genes from Eucalyptus were performed. The results suggest that the HD-Zip family can be divided into four subfamilies, HD-ZipI-IV [24]. Multiple sequence alignments of and phylogenetic tree showed a high homology among these genes. Conserved domain analysis suggests that all EgHD-Zip proteins except EgHD-Zip30 have motifs 1, 2, and 5. SMART analysis indicates that these three motifs represent the HD domain and LZ domain. The study of subfamily III and subfamily IV showed that both have motifs encoding HD, LZ, START, and SAD domains. In contrast, subfamily IV does not have motifs 8 and 4 that encode MEKHLA domains. Previous studies have shown that subfamily III is found in terrestrial plants. It is also worth noting that the MEKHLA domain was formed before the emergence of terrestrial plants, which could be the "preliminary preparation" for plants to adapt the terrestrial living conditions. Therefore, it can be speculated that subfamilies III and IV have the most recent comment ancestor. The analysis of gene structure showed that the exon-intron arrangement of different subfamilies has significant differences, while the same subfamily has similar structures and exon-intron numbers. But in the evolution process, exons would be lost or increased, and the exon length of each gene may also change. We found that HD-Zip35 lost an intron and HD-Zip7 and HD-Zip6 obtained an intron during the evolution, which could be one of the factors affecting the different functions of homologous genes.
The expression heat map analysis showed that the subfamily I has highly expressed, or even the highest expressed genes in the five tissues studied. It is speculated that they may be playing a significant role in the growth and development of Eucalyptus. In addition to  The position data is displayed on the right side of the chromosome, and a short line connects the corresponding EgHD-Zip gene names on the left side, and different colors represent different subfamilies. Black is the EgHD-Zip I subfamily, red is the EgHD-Zip II subfamily, green is the EgHD-Zip III subfamily, blue is the EgHD-Zip IV subfamily. Chr, chromosome the low expression level of subfamily II in mature leaves, the other four tissues have high expression genes, indicating that this family plays an essential role in the growth and transport of Eucalyptus. In the III subfamily, all genes are expressed in vascular bundle tissues, with high expression levels, but low expression in leaf and stem tips. Therefore, it can be speculated that they play an important role in Eucalyptus auxin transport and vascular tissue formation.
Interestingly, the expressions of the IV and III subfamilies were opposite in the five selected tissues. It is worth noting that HD-Zip38 was not expressed in the five selected samples, which does not mean that it is not important in the growth and development of Eucalyptus. On the contrary, it may be expressed explicitly in other tissues, thereby affecting the growth and development of Eucalyptus (Fig. S3, S4, S5, S6, S7).
The expression of HD-Zip in the young leaves treated with distilled water showed a trend of first decreasing and then increasing. After 6 h under salt stress, the EgHD-Zip gene showed some physiological activity, the expression first increased, then it dropped, followed by an increase again with the lowest expression level after 12 h of stress. It can be speculated that under moderate salt stress, the expression of HD-Zip gene is suppressed in young leaves of Eucalyptus.
The expression pattern from two selected genes, EgHD-Zip27 from HD-ZipII subfamily and EgHD-Zip37 from HD-ZipI subfamily, suggest that both the subfamilies play an essential role in coping with salt stress, and play a role in the growth and development of leaves (Fig. 9).  6 Relative expression levels of EgHD-Zip genes in various tissues. Expression profiles of EgHD-Zip genes in young leaf, xylem, shoot tips, phloem, mature leaf and immature xylem of Eucalyptus. Red indicates that the gene is highly expressed in this tissue, and blue indicates low expression. The color scale is shown on the right side of the graph. The prefix "Eg" stands for Eucalyptus grandis Under low-temperature stress, the expression pattern of HD-Zip gene mostly decreased first, then increased and decreased again. Under high-temperature stress, the expression of HD-Zip27 was the same as that under 25°C treatment. At the same time, HD-Zip37 shows a pattern of first decreasing, then increasing and then decreasing again, suggesting its regulation by temperature change.

Conclusion
HD-Zip family plays a vital role in the growth and development of plants. Here, a total of 40 EgHD-Zip genes were identified in Eucalyptus. Based on the evolutionary analysis, the EgHD-Zip family of Eucalyptus was divided into four groups. Moreover, genes that are in the same group tend to have similar exon-intron structures, motifs, and protein structures. During exposure to salt and temperature stresses, the expression of HD-Zip genes in Eucalyptus show differential expression. We here show that HD-Zip may play a unique role under different stress conditions. This study provides a basis for further research on the functional characterization of the Eucalyptus HD-Zip genes. It also provides a theoretical basis for other scholars to study the response of the Eucalyptus HD-Zip transcription factor during abiotic stress. As the land salinization is becoming a severe threat, further research for the improvement of Eucalyptus varieties could help in the expansion of Eucalyptus planting zones.

Methods
Eucalyptus HD-zip transcription factor family member: identification and chromosomal localization HD-Zip protein sequences from Arabidopsis and rice were used as query in the TAIR database (http://www.arabidopsis. org) and PlantTFDB v4.0 database (Plant Transcription Factor Database). Also, the Eucalyptus HD-Zip HMM search [66] was carried out to integrate all the protein sequences obtained, in conjunction with SMART (Simple Molecular Agricultural Research Tool, http://smart.embl-heidelberg.de/ ) and EMBL pfam (https://pfam.xfam.org/) database was used to detect the conserved domains. Finally, the wrong sequence was removed by manual screening. The 40 candidate sequences after the screenings were considered to be the Eucalyptus HD-Zip gene (EgHD-Zip). The chromosomal locations of the EgHD-Zip genes were obtained from the Fig. 7 RT-PCR expression profiles of five subfamilies of genes in HD-Zip in response to salt stress. The RNA was isolated from the young leaves of the treated Eucalyptus annual seedlings. The low salinity was treated with 100 mmol/L NaCl, the high salinity was treated with 200 mmol/L NaCl, and the control group was treated with distilled water. The processing time of each group was 0 h, 6 h, 12 h and 24 h genomic annotation information of phytozome v12.1.6 (https://phytozome.jgi.doe.gov/pz/portal.html), and using the Mapchart software it was visualized.

HD-Zips
Multiple sequence alignments of Eucalyptus, rice and Arabidopsis HD-Zip protein sequences were performed using MUSCLE v3.8.31 [67]. The IQ-Tree software was built using the Maximum Likelihood (ML) method to construct a phylogenetic tree with the bootstrap option n = 1000. After that, the results were imported to iTOL (https://itol.embl.de/) online software for processing.

Protein conserved motif and gene structure analysis
The Gene Structure Display Server (GSDS) [68,69] was used to display the exon-intron of the Eucalyptus HD-Zip gene. We downloaded the annotation file in the format of GFF from Phytozome and retrieved the annotation information of the HD-Zip gene. The annotation information was uploaded to GSDS according to the default parameters of the exon-intron structure of the HD-Zip gene. Multiple Em for Motif Elicitation (MEME) was used to further determine the composition of motifs that might not be recorded in the public database [70,71]. The width was kept 100aa, the number of motifs was 25, and other parameters were set to default values. Finally, based on the protein homology model in the swiss model database, we also inferred the 3D structure of the HD-Zip protein of Eucalyptus.

Analysis of EgHD-Zip expression pattern
RNA-Seq data were downloaded from public websites and Phytozome [72]. Among them, the Eucalyptus RNA-Seq data included the following tissues: immature xylem, mature leaf, phloem, shoot tips, xylem. The heat maps of EgHD-Zip expression profiles from different tissues were obtained using TBtools software [73].

Plant materials and growth conditions
The experimental material, Eucalyptus grandis clone Eg5, was collected from the College of Forestry, Fujian Agriculture and Forestry University. Eucalyptus grandis plants were grown on local soil for ten months under Fig. 8 RT-PCR expression profiles of five subfamilies of genes in HD-Zip in response to temperature stress. The RNA was isolated from the young leaves of the treated Eucalyptus annual seedlings. The low temperature was treated with 4°C, the high temperature was treated with 40°C, and the control group was treated with 25°C. The processing time of each group was 0 h, 6 h, 12 h and 24 h outdoor conditions. The annual average temperature in the growing area ranged from 16 to 20°C, the yearly average rainfall from 900 to 2100 mm, and the annual relative humidity was about 77%. The red soil, which has the organic matter content from 2.57 to 6.07%, and the pH value 5, was used for cultivation.

Expression profile of EgHD-Zip gene under temperature and salt stress
Annual Eucalyptus seedlings were treated with 100 mmol/L sodium chloride and 200 mmol/L sodium chloride for 0, 6, 12, and 24 h for salt stress, and the control seedlings were treated with distilled water. For the temperature stress, Eucalyptus seedlings were placed at 4°C and 40°C, respectively, while and the control group was kept at room temperature (at 25°C) for 0, 6, 12, and 24 h. After the stress, the samples were stored at − 80°C for the subsequent analysis. For each treatment, five different seedlings were used, and the experiment was repeated at least three times.

Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted using RNA extraction Kit (Omega Bio-Tek, Shanghai, China) from control and stress treated samples. cDNA was synthesized with the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix following the manufacturer's protocol (Transgen, Beijing, China).and qRT-qPCR was conducted using TransStart® Top Green qPCR SuperMix (Transgen, Beijing, China). As a reference gene actin was used. Relative transcript abundance was calculated using the comparative 2 -ΔΔC T method [74]. All experiments were performed using three biological replicates and three technical replicates. The primers used in the study are listed in the supplementary Table S1.

Statistical analysis
A two-tailed Student's t-test was applied to find statistical significance. Results are depicted as the mean values ± SE of three biological replicates. Fig. 9 Working model of HD-Zip during developmental and stress (salt) response. The unknown receptor present on the plasma membrane perceives the stress or growth signal. Phytohormone, stress, or a change in the concentration of cytosolic Ca 2+ may serve as the signal for the expression of HD-Zip transcription factors, which in turn regulate the expression of target genes resulting in growth, development and stress tolerance. In the case of salt exposure, the Na + enters inside non-selective cation channels (NSCC) and some other transporters. Change in the concentration of cytosolic Ca 2+ activates the CBL-CIPK signaling pathway leading to the increased activity of Na + /H + antiporter SOS1 (salt overly sensitive1), which transport the Na + in the vacuole or outside the cell. Phytohormone ABA levels also regulate the Na + /H + antiporter NHX1 and other ion transporters to overcome with stress